Project Summary. SARS-CoV-2, or the 2019 novel coronavirus, is a significant pandemic threat that has resulted in hundreds of thousands of diagnosed cases and tens of thousands of mortalities as of March 2020. The development of preventive and therapeutic measures that can counteract the ongoing, and any future, coronavirus pandemics is therefore of utmost significance for public health worldwide. The S protein is the immunodominant region of coronaviruses (CoV) recognized by the immune system and serves as the target for a number of neutralizing antibodies. Passive transfer of neutralizing antibodies has been shown to prevent coronavirus infection in animal models. Further, engineered prefusion-stabilized S protein immunogens have been shown to elicit high titers of coronavirus-neutralizing antibodies in animal models, in the context of MERS. Together, this prior work establishes a strong premise for targeting the identification and characterization of neutralizing antibodies in the context of SARS-CoV-2. More generally, a better understanding of the human antibody response to the S protein of SARS-CoV-2 as well as other related CoV members can help inform therapeutic antibody optimization and accelerate vaccine design efforts. Our laboratory recently developed the LIBRA-seq technology (LInking B-cell Receptor to Antigen specificity through sequencing) for antibody discovery and characterization of antigen-specific antibody repertoires. Unlike other B cell approaches, LIBRA-seq is the first to enable the simultaneous determination of BCR sequence and antigen specificity for a large number of B cells against a theoretically unlimited number of diverse antigens, at the single-cell level. LIBRA-seq therefore provides a unique opportunity for characterizing the types and specificities of antibodies that can recognize the S protein from SARS-CoV-2, as well as other CoV viruses. Here, we propose to utilize the LIBRA-seq technology in the context of SARS-CoV-2, with two major goals: (1) To identify cross-reactive antibodies that recognize multiple antigen variants associated with human coronavirus infection, including SARS-CoV-2, SARS-CoV-1, and MERS-CoV, and (2) To evaluate the ability of current lead CoV vaccine candidates to engage with antibody repertoires from healthy individuals. Taken together, the efforts proposed in this application will be of high potential translational/clinical impact for SARS-CoV-2 and other CoV pathogens of biomedical significance. The types of antibody repertoire characterization that we propose to develop here will also be readily generalizable to other pathogens, and as such, will have a broad and lasting impact on the development of countermeasures for established and emerging infectious diseases.